Synthetic deoxygenated derivatives of methyl beta-glycosides of (1----6)-beta-D-galacto-oligosaccharides were prepared, and their binding to antigalactan monoclonal antibodies X24 and J539 (Fab') was studied. The results suggest the involvement of an additional, critical hydrogen bond in the highest affinity subsite (A), which now appears to require two hydrogen bonds from the 2- and 3-hydroxyls of the galactosyl residue to the protein, and one from the protein to O-4 of that residue. The data obtained with a series of oligosaccharides deoxygenated at position 3(1), 3(2), 3(3), 4(1), 4(2), or 4(3) support the binding patterns and subsite-arrangement inferred previously from studies with large numbers of deoxyfluoro-substituted ligands and this family of antibodies.